The value of AR-V7 in predicting therapeutic efficiency of abiraterone for CRPC patients

Objective To investigate the predictive effect of androgen receptor splice variant 7 (AR-V7) expression on time to castration resistance in patients with metastatic prostate cancer.Methods The data of 113 cases of advanced metastatic prostate cancer diagnosed by prostate biopsy in our institute from Jan.2002 to Jun.2010 were collected retrospectively.The median age at diagnosis was 70 years,ranged from 43 to 84 years.The median tPSA was 120.0 μg/L,ranged from 3.0 to 6 006.2 μg/L.There were 5 patients in M1a(4.4％),94 patients in M1b(83.2％) and 14 patients in M1c(12.4％).All patients received hormonal therapy.1mmunohistochemical staining and AR-V7 specific antibody were used to detect the expression of AR-V7 in prostate cancer tissues.Cox regression models were used to analyze the predictive role of patient characteristics including patient's age at diagnosis,tPSA level at diagnosis,Gleason score,clinical stage,PSA nadir during hormonal therapy,the time to PSA nadir and PSA half-life.The effect of AR-V7 expression on time to castration resistance was analyzed using Kaplan-Meier curves,and the differences were assessed using the log-rank test.Results The PSA nadir ranged from 0.0 to 143.0 μg/L and the median value was 0.7 μg/L.The time to PSA nadir ranged from 0.9 to 71.0 months and the median value was 8.1 months.The median PSA half life was 1.0 month,which ranged from 0.1 to 41.0 months.Followed up for a median time of 27 months,100 patients progressed to castration-resistant prostate cancer (CRPC) and the median time to castration resistance was 24 months.The expression of AR-V7 was positive in 23 out of 113 patients.Multivariate analysis showed that the expression of AR-V7 in prostate cancer (P=0.004,HR =2.223) and PSA nadir during hormonal therapy (P =0.035,HR =1.011) were independent predictive factors of time to castration resistance.The median time to castration resistance for patients with and without AR-V7 expression were (16.04±3.4) months and (30.04-6.0) months,respectively (P=0.001).Conclusions The expression of AR-V7 in prostate cancer and PSA nadir during hormonal therapy are independent predictive factors of time to castration resistance.The AR-V7 could be a potential therapeutic target for advanced prostate cancer. Key words: Prostatic neoplasms; Hormonal therapy; Time to castration resistance; Androgen receptor splice variant 7; PSA nadir

Background: The androgen receptor (AR) is a clinically important driver in prostate cancer. In metastatic castration resistant prostate cancer (mCRPC), increased expression of the ligand-independent AR variant 7 (AR-v7) is a biomarker of hormonal therapy resistance. However, the prevalence and clinical importance of AR-v7 in non-metastatic CRPC (nmCRPC) is not yet known. Low circulating tumor cell (CTC) frequencies in these patients make emergence of AR-v7 during anti-androgen therapy difficult to study. We report blood-based detection of AR-v7 using digital droplet PCR (ddPCR) in nmCRPC patients enrolled in the SPARTAN trial, a randomized phase 3 study testing ADT vs apalutamide (APA)+ADT. Method: To investigate simultaneous and quantitative expression of AR-v7 and AR, we utilized ddPCR to measure individual mRNA transcripts in blood samples collected in PAXgene tubes. AR-v7 positivity was calculated as the normalized fraction of AR-v7 vs total AR transcripts (AR-v7/AR). Normalized AR-v7/AR frequency in healthy volunteers (HV) and mCRPC was measured to determine an expression cutoff to separate normal and prostate cancer blood samples. The ddPCR AR-v7 biomarker assay was then used to measure AR-v7 expression in ADT (N=47) and APA+ADT (N=53) SPARTAN samples taken at time of study initiation and correlated with clinical outcome. Results: By setting a cutoff at 0.3 AR-v7/AR normalized fraction we could differentiate HV from mCRPC patients. In nmCRPC, mean AR-v7 and AR-FL expression were calculated as 1.2 and 349.3 transcripts, respectively. The 0.3 AR-v7/AR normalized fraction cutoff could not differentiate AR-v7 expression between HV and nmCRPC. Using this assay, we detected AR-v7 transcripts in 47% of nmCRPC SPARTAN patients analyzed. However, results of AR-v7 expression as a continuous and discretized variable were inconclusive when correlated with clinical outcome. Conclusion: This study reports ddPCR-based detection of whole blood mRNA as a sensitive assay to detect simultaneously low and high expressing AR transcripts in nmCRPC. Our technical analysis demonstrates that unlike in mCRPC, low level transcript counts of AR-v7 in nmCRPC may not distinguish expression from baseline in healthy patients. Data from this limited cohort suggest that while AR-v7 is detected in 47% of patients, a higher threshold of expression may be biologically important for driving treatment resistance. Further analysis of this assay in mCRPC and APA refractory samples sequenced with other therapies are needed to confirm the clinical and biological utility of AR-v7 detection by ddPCR assay and inform disease continuum management. Citation Format: Mel Pilar Espaillat, Yashoda Rajpurohit, Mike Gormley, Denis Smirnov, Ian McCaffery, Angela Lopez-Gitlitz, Deborah Ricci, Shibu Thomas. Digital droplet PCR (ddPCR)-based detection of androgen receptor splice variant 7 (AR-v7) in non-metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1335.

Background: The androgen receptor (AR) is a clinically important driver in prostate cancer. In metastatic castration resistant prostate cancer (mCRPC), increased expression of the ligand-independent AR variant 7 (AR-v7) is a biomarker of hormonal therapy resistance. However, the prevalence and clinical importance of AR-v7 in non-metastatic CRPC (nmCRPC) is not yet known. Low circulating tumor cell (CTC) frequencies in these patients make emergence of AR-v7 during anti-androgen therapy difficult to study. We report blood-based detection of AR-v7 using digital droplet PCR (ddPCR) in nmCRPC patients enrolled in the SPARTAN trial, a randomized phase 3 study testing ADT vs apalutamide (APA)+ADT. Method: To investigate simultaneous and quantitative expression of AR-v7 and AR, we utilized ddPCR to measure individual mRNA transcripts in blood samples collected in PAXgene tubes. AR-v7 positivity was calculated as the normalized fraction of AR-v7 vs total AR transcripts (AR-v7/AR). Normalized AR-v7/AR frequency in healthy volunteers (HV) and mCRPC was measured to determine an expression cutoff to separate normal and prostate cancer blood samples. The ddPCR AR-v7 biomarker assay was then used to measure AR-v7 expression in ADT (N=47) and APA+ADT (N=53) SPARTAN samples taken at time of study initiation and correlated with clinical outcome. Results: By setting a cutoff at 0.3 AR-v7/AR normalized fraction we could differentiate HV from mCRPC patients. In nmCRPC, mean AR-v7 and AR-FL expression were calculated as 1.2 and 349.3 transcripts, respectively. The 0.3 AR-v7/AR normalized fraction cutoff could not differentiate AR-v7 expression between HV and nmCRPC. Using this assay, we detected AR-v7 transcripts in 47% of nmCRPC SPARTAN patients analyzed. However, results of AR-v7 expression as a continuous and discretized variable were inconclusive when correlated with clinical outcome. Conclusion: This study reports ddPCR-based detection of whole blood mRNA as a sensitive assay to detect simultaneously low and high expressing AR transcripts in nmCRPC. Our technical analysis demonstrates that unlike in mCRPC, low level transcript counts of AR-v7 in nmCRPC may not distinguish expression from baseline in healthy patients. Data from this limited cohort suggest that while AR-v7 is detected in 47% of patients, a higher threshold of expression may be biologically important for driving treatment resistance. Further analysis of this assay in mCRPC and APA refractory samples sequenced with other therapies are needed to confirm the clinical and biological utility of AR-v7 detection by ddPCR assay and inform disease continuum management. Citation Format: Mel Pilar Espaillat, Yashoda Rajpurohit, Mike Gormley, Denis Smirnov, Ian McCaffery, Angela Lopez-Gitlitz, Deborah Ricci, Shibu Thomas. Digital droplet PCR (ddPCR)-based detection of androgen receptor splice variant 7 (AR-v7) in non-metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1335.

To the Editor: There are no data regarding expressed and functional characterisation of cytoskeleton-related non-coding RNA has been reported in prostate cancer (PCa). Here, we report a cytoskeleton regulator RNA (CYTOR)-regulated process that mediates castration-resistant PCa (CRPC)-specific androgen receptor splice variant 7 (AR-V7) generation, and further explore the vulnerability of CRPC growth through CYTOR-targeted locked nucleic acid (LNA). We retrieved public castration-sensitive PCa (CSPC) datasets (n = 65), neuroendocrine PCa (NEPC) datasets (n = 49) and CRPC datasets (including two studies, n = 171 and n = 118).1 Across the above RNA-seq data, CYTOR was found to be upregulated in CRPC with low expression in CSPC and NEPC (Figure 1A). RNA in situ hybridisation (RISH) assays2 of our centre samples confirmed the public domain data (Figure 1B and Figure S1A). Consistent with tissue detection, androgen-influenced CYTOR revealed significant increase in our two continuous established castration-resistant cell lines: LNCaP-AI,3 C4-2 Enz-R (Figure 1C and Figure S1B–G). Additionally, progression was more common in CSPC with higher CYTOR expression (Figure 1D). Expression analysis of CYTOR in flash-frozen surgical specimens was conducted in 11 CRPC patients (Figure 1E). Patients with high expression of CYTOR received worse PSA response to subsequential enzalutamide than those with CYTOR low expression (Figure 1F). Then, gene functional assays suggested knockdown of CYTOR suppressed the cancer cells growth (Figure 1G–J). The above results hint the association of CYTOR with CRPC development and inferior clinical outcomes. As it is, the primary therapeutic intervention for advanced PCa is androgen-deprivation therapy (ADT) with the goal of castration to suppress androgen receptor (AR) signalling. Although most patients respond to ADT, some inevitably develop resistance and progress to CRPC because of AR-V7 expression.4 Extensively investigated AR-V7 is a typically truncated AR without the ligand-binding domain but retaining transcriptional-regulated activity to mediate ligand-independent AR signalling.5 RNA-seq analysis revealed many AR-V7 downstream genes were differentially regulated as CYTOR knockdown (Figure 2A, Table S2). Most of them were enriched in metabolic pathways (Figure 2B). We validated the downregulation of AR-V7 canonic-activated genes (Figure 2C) after silencing CYTOR. Interestingly, knockdown of CYTOR resulted in specific decrease of AR-V7 without concurrent decrease of full-length AR (AR-FL) (Figure 2D), suggesting the critical role of CYTOR in AR-V7 mRNA splicing process. Multiplexed RISH assays of CRPC specimens revealed colocalisation and positive correlation of CYTOR and AR-V7 (pre-mRNA accumulated in nuclei) (Figure 2E). Their positive correlation was also confirmed by RT-PCR in four flash-frozen specimens (Figure 2F). Because key RNA-binding protein families involved in alternative splicing may include serine/arginine-rich proteins (SR proteins) and heterogeneous nuclear ribonucleoproteins (hnRNPs), we conducted differential expression analysis of SR proteins and hnRNPs between LNCaP-AI and LNCaP cells by our published RNA-arrays (GSE124291), and screened six upregulated splicing factors in LNCaP-AI cells (>1.5-fold) (Figure 2G). We further confirmed the upregulation of three genes (Figure 2H and Figure S2A). By Human Splicing Finder,6 the similar consensus splice site value for splice junctions of intron 3/cryptic exon 3 (CE3) (80.38) (as in AR-V7) and intron 3/exon 4 (80.1) (as in AR-FL) (Figure S2B) suggested the existence of a mechanism for CRPC-specific CE3 splice site utilisation. Given the established role of SR proteins in binding to pre-mRNA that prevents exon skipping, and the classical role of hnRNPs as splicing repressors, we postulated that nuclear-localised SRSF4 and SRSF7 (Figure 2I) may repress CE3 skipping, thus ensuring the correct 5′ to 3′ linear order of exons (exon1-3/CE3) in AR-V7 mRNA. Indeed, knockdown of SRSF4 or SRSF7 resulted in decreased expression of AR-FL and AR-V7, while withoutimpact on CYTOR (Figure 2J). The catRAPID strength algorithm computed output suggested the high specificity of CYTOR–SRSF4 interaction and CYTOR–SRSF7 interaction, respectively (Figure 2K,L).7 RNA immunoprecipitation results revealed both SRSF4 and SRSF7 proteins interacted with CYTOR, AR-V7 pre-mRNA and AR-V7 mRNA (Figure 2K,L), indicating nuclear binding of SRSF4 and SRSF7 to AR-V7 pre-mRNA and CYTOR was responsible for AR-V7 generation, even though there was weak interaction of SRSF4 and SRSF7 (Figure 2M). According to the functional interaction of CYTOR and SRSF4/7 proteins, we hypothesised that CYTOR may recognise AR-V7 pre-mRNA to induce this splicing process. Toward this end, maximum entropy modeling was used to collect motifs in the intron3/CE3 flanking sequence and identified the 3′ motif (3′ site of intron 3) and the 5′ motif (first 20 bp of CE3) (Figure 3A–C).6 The complementary sequence of the 5′ motif in the sequence of CYTOR (5′-UUCCAACCGC-3′) suggested that CYTOR may recognise the 5′ motif of CE3 (5′-GGGUUGGCAA-3′) to initiate the splicing process (Figure 3C). Next, we designed an 18 bp antisense oligonucleotides (ASO) to the 5′ motif of CE3 (ASOCE3) to prevent the recognition. The ASOCE3 suppressed, in a concentration- and time-dependent manner, the expression of AR-V7 mRNA (Figure 3D). We then designed an 18 bp ASOCYTOR to the complementary sequence of CE3 5′ motif in CYTOR. ASOCYTOR inhibited expression of AR-V7 mRNA without interfering CYTOR expression (Figure 3E and Figure S2C). Also as shown in C4-2 Enz-R cells, the ASOCE3 and ASOCYTOR prevented the generation of AR-V7 mRNA (Figure 3F,G). To validate this splicing model, truncated mutant assays confirmed the pivotal role of 5′-UUCCAACCGC-3′ in CYTOR on AR-V7 expression (Figure S2D,E). Together, CYTOR/SRSF4/SRSF7 complex interacts with AR-V7 pre-mRNA to regulate its splicing by recognising a specific signal element in CE3 (Figure 3H). Then LNAs GapmeRCYTOR were designed to silence CYTOR (Figure 4A). In C4-2 Enz-R cells, AR-V7 expression was largely suppressed in parallel with the silenced pattern of CYTOR in a concentration- and time-dependent manner (Figure 4B–E). GapmeRCYTOR could attenuate the resistance of enzalutamide significantly in vitro (Figure 4F). We then established in vivo mouse models and found that enzalutamide significantly suppressed C4-2 tumours, shCYTOR- and GapmeRCYTOR-treated C4-2 Enz-R tumours (Figure 4G,H). The expressions of CYTOR and AR-V7 were validated in each group (Figure S2F,G). As such, our data suggested that on-target effect of CYTOR knockdown with GapmeRCYTOR can be used as an option in castration resistance to provide therapeutic efficacy. In conclusion, we propose the importance of a novel complex composed of CYTOR/SRSF4/SRSF7 that mediates AR-V7 generation, and a critical role in suppressing PCa progression by targeting CYTOR/AR-V7 axis with shRNA or preclinical LNA GapmerCYTOR. Some of the biospecimens used in the present study were provided by the Chungbuk National University Hospital, a member of the National Biobank of Korea, which is supported by the Ministry of Health, Welfare, and Family Affairs. All samples derived from the National Biobank of Korea were obtained with informed consent under institutional review board-approved protocols. The authors wish to thank Ms. Eun-Ju Shim from the National Biobank of Korea at Chungbuk National University Hospital for the sample preparations and her excellent technical assistance. The authors declare they have no conflicts of interest. National Natural Science Foundation of China, Grant Numbers: 91959114, 81872106, 82072851, 81872100, 81972654, 82273262; National Natural Science Foundation of China, International (Regional) Cooperation and Exchange Program, Grant Number: 82061160493; Natural Science Foundation of Tianjin, Grant Numbers: 18PTLCSY00030, 21JCQNJC01700; Tianjin Key Medical Discipline (Specialty) Construction Project, Grant Numbers: TJYXZDXK-023A, TJYXZDXK-065B; The Second Hospital of Tianjin Medical University, Grant Number: 2020ydey01; Scientific Research Project of Tianjin Education Commission, Grant Number: 2021KJ225 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

In clinical samples, the expression of androgen receptor (AR) and of AR splice variant 7 (AR-V7) is higher in castration-resistant prostate cancer (CRPC) compared with that in hormone-sensitive prostate cancer (PCa). However, there are only a few reports on the ratio of the expression levels of AR-V7 to AR (AR-V7/AR) in prostate tissue. The present study evaluated AR-V7/AR expression in various types of human prostate tissues and CRPC cells. Pretreatment prostate tissue samples from patients with benign prostatic hyperplasia (BPH; n=18), Gleason score 7 (n=17), and Gleason score 8–10 (n=26) were collected at the time of prostate biopsy, and tissue samples from CRPC patients (n=10) were collected at the time of transurethral resection of the prostate. Furthermore, androgen-independent LNCaP cells were established. The mRNA expression levels of AR and AR-V7, cell proliferation and prostate-specific antigen (PSA) production were evaluated by reverse transcription quantitative PCR, MTS assay and chemiluminescent enzyme immunoassay, respectively. There was a significant difference in AR-V7/AR expression ratios between the CRPC group and the BPH and pre-treatment PCa groups (CRPC, 7%; BPH and pre-treatment PCa, 1%). Subsequently, we compared the AR and AR-V7 expression levels in CRPC samples with those in the pretreatment prostate tissues from the same patients. The results demonstrated that the AR-V7/AR ratio increased from 3 to 9% after CRPC onset. Furthermore, in vitro experiment demonstrated that AR-V7 expression in LNCaP cells was increased after transforming into CRPC cells. The AR-V7/AR ratio also increased from 0.05 to 0.3%. In addition, small interfering (si)-RNA-mediated knockdown of AR inhibited the proliferation of and PSA production from androgen-independent LNCaP cells; however, AR-V7 knockdown had no effect. Conversely, siRNA-mediated knockdown of both AR and AR-V7 inhibited the proliferation of VCAP cells. In summary, the findings from the present study demonstrated that AR-V7 expression and AR-V7/AR ratio were increased after the onset of CRPC, which had a limited role in CRPC cell proliferation. Further investigation is required to clarify the roles of AR other splice variants and AR-V7 in CRPC.

Exploring the association between metastatic sites and androgen receptor splice variant 7 (AR-V7) in castration-resistant prostate cancer patients: A meta-analysis of prospective clinical trials

BACKGROUND: The androgen-receptor splice variant 7 (AR-V7) has been implicated in the development of castration resistant prostate cancer (CRPC) and resistance to current therapies including enzalutamide and abiraterone. AR-V7 mRNA expression in circulating tumour cells of patients with CRPC correlated with treatment resistance. However, the importance of AR-V7 has been questioned in light of low AR-V7 mRNA levels relative to the full-length androgen receptor in CRPC and it is critically important to develop validated assays that confirm AR-V7 protein levels and its clinical importance in patients with CRPC. METHODS: Following validation of a monoclonal antibody, immunohistochemical analysis of nuclear AR-V7 (alongside a nuclear AR N-terminal domain antibody; AR-NTD) was performed in a patient cohort identified with matched therapy-naive hormone-sensitive primary prostate cancer (HSPC) and CRPC. We determined the levels of nuclear AR-V7 as patients progressed from HSPC to CRPC. We also determined if AR-V7 expression levels associated with overall survival from time of CRPC biopsy. RESULTS: In our patient cohort (n = 39), nuclear AR-V7 (p = <0.0001) and nuclear AR-NTD (p = 0.0006) increased significantly as patients progressed from HSPC to CRPC. Lower nuclear AR-V7 expression was associated with improved overall survival from time of CRPC biopsy in patient groups divided by the 25th (18.7 vs 9.6 months; HR 0.36 [95% CI 0.17-0.62]; p = 0.002), 50th (13.0 vs 9.8 months; HR 0.60 [95% CI 0.29-1.07]; p = 0.09) or 75th (6.0 vs 11.5 months; HR 0.31 [95% CI 0.04-0.34]; p = 0.0004) percentile of AR-V7 expression. Similarly, a lower nuclear AR-V7 to nuclear AR-NTD ratio was associated with improved overall survival from time of CRPC biopsy in patient groups divided by the 25th (17.8 vs 9.1 months; HR 0.42 [95% CI 0.22-0.78], p = 0.01), 50th (14.0 vs 8.8 months; HR 0.43 [95% CI 0.17-0.69], p = 0.005) and 75th (11.5 vs 7.3 months; HR 0.39 [95% CI 0.09-0.69], p = 0.009) percentile. Nuclear AR-NTD did not associate with overall survival from CRPC biopsy. CONCLUSION: We provide first evidence that expression of nuclear AR-V7 protein not only increases with emerging treatment resistance in CRPC but also is associated with overall survival from time of CRPC biopsy. These data support AR-V7 protein being key to CRPC progression and that agents targeting AR splice variants will be important to improve patient outcome in CRPC. Citation Format: Daniel Nava Rodrigues, Jon Welti, Adam Sharp, Shihua Sun, Ruth Riisnaes, Ines Figueiredo, Zafeiris Zafeiriou, Pasquale Rescigno, Johann S. de Bono, Stephen R. Plymate. Analytic validation and clinical qualification of a novel immunohistochemical assay for AR-V7 protein expression in metastatic prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4965.

AR-V7 in Peripheral Whole Blood of Patients with Castration-resistant Prostate Cancer: Association with Treatment-specific Outcome Under Abiraterone and Enzalutamide

Androgen Receptor Modulation Optimized for Response—Splice Variant: A Phase 3, Randomized Trial of Galeterone Versus Enzalutamide in Androgen Receptor Splice Variant-7–expressing Metastatic Castration-resistant Prostate Cancer

Prostate Cancer (PCa) is the third most common cause of death from cancer in men of all ages. Surgical or medical castration is one of the most common treatments for patients with advanced PCa; however a majority of patients develop castration resistant prostate cancer (CRPC), tumor relapse, which remains to be the second leading cause of cancer-related deaths of men in the US. Androgen receptor (AR) signaling is shown to play a critical role in the development and progression of PCa. Genetic aberrations within AR, including constitutively active AR splice variants and AR point mutations have been identified in CRPC. The most common AR splice variants lack the ligand-binding domain (LBD), which is often the target of CRPC therapies. Therefore, presence of these variants may act as a mechanism of resistance to AR-targeted therapies leading to the progression of prostate tumor growth. Additional PCa specific genetic aberrations include fusions between the androgen-related gene, TMPRSS2 and the ETS transcription factors, ERG (predominant) and ETV1. These fusion events are frequently associated with more aggressive prostate cancers leading to poorer prognosis. In this study, we developed TaqMan qRT-PCR assays to evaluate the presence of several previously identified AR splice variants, including ARV1, ARV3/V7, ARV567 and ARV8, AR somatic mutations, including L701H, V715M, H874Y and T877A, along with TMPRSS2 fusion genes, TMPRSS2:ERG and TMPRSS2:ETV1, in two independent PCa FFPET sample sets. The first sample set consisted of 42 Prostate adenocarcinomas ranging from stage II to stage IV. Results showed that ARV1 and ARV3/V7 were the most prevalent variants with 92% of all samples showing expression of either or both variant. TMPRSS2: ERG was present in 72% of all samples tested, with a high concordance to AR variant expression, prevalent in later stage (III/IV) PCa samples. The second sample set consisted of 8 prostate adenocarcinomas, including matched adjacent normal FFPET. Similar expression of the AR variants was observed in both the tumor and matched normal samples, however tumor prostate samples showed a higher and more prevalent expression (66.67%) of the TMPRSS2: ERG fusion gene than in the matched normal samples (33%). None of the four AR mutations evaluated were detected in either sample set. Overall, these findings demonstrate a strong presence of both AR splice variants and the TMPRSS2: ERG fusion gene in the prostate cancer patient population, supporting evidence for a functional role of these markers in PCa diagnosis and disease progression. Furthermore, presence of LBD negative AR splice variants indicates an attractive biomarker for stratification of the patient population resistant to AR targeted therapies. Citation Format: Dana S. Gaffney, Gabriela Martinez, Katherine Bell, Suso Platero, Deborah Ricci, Jayaprakash Karkera. Identification of androgen receptor (AR) splice variants, AR somatic mutations and TMPRSS2:ETS fusion genes in prostate cancer FFPET by qRT-PCR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5. doi:10.1158/1538-7445.AM2013-5

Prognostic Value of Androgen Receptor Splice Variant 7 in the Treatment of Castration-resistant Prostate Cancer with Next generation Androgen Receptor Signal Inhibition: A Systematic Review and Meta-analysis

Identification of AR-V7 downstream genes commonly targeted by AR/AR-V7 and specifically targeted by AR-V7 in castration resistant prostate cancer

<div>Abstract<p><b>Purpose:</b> Despite the efficacy of abiraterone, a CYP17A1 inhibitor, in metastatic castration-resistant prostate cancer (CRPC), nearly all patients develop resistance. The purpose of this phase II study was to evaluate mechanisms of resistance to more complete androgen synthesis inhibition with abiraterone and dutasteride.</p><p><b>Experimental Design:</b> Eligible patients with metastatic CRPC underwent a baseline metastasis biopsy. Patients received abiraterone and prednisone for two 4-week cycles. After this time, high-dose dutasteride (3.5 mg daily) was added. Patients continued therapy until study withdrawal or radiographic progression. Repeat metastasis biopsy was obtained at progression. The primary endpoint was to assess mechanisms of resistance. Serum hormone and abiraterone levels were assessed. Tissue was assessed for androgen receptor (AR) and AR splice variant-7 (ARV7) expression.</p><p><b>Results:</b> Forty patients were enrolled. Sixty percent (<i>n</i> = 24) achieved a ≥50% reduction in prostate-specific antigen (PSA). The median time to radiographic progression was 11 months. Nearly all baseline (<i>n</i> = 29 of 31) and posttreatment (<i>n</i> = 16 of 16) tumors tested for AR nuclear expression were positive. Of those tested, ARV7 expression was present in 48% (<i>n</i> = 10 of 21) of baseline and 42% (<i>n</i> = 5 of 12) of treatment discontinuation specimens. Compared with patients with higher serum abiraterone levels at treatment discontinuation, patients with lower levels had higher circulating androgens.</p><p><b>Conclusions:</b> Despite increased androgen synthesis inhibition, we demonstrate that tumor AR axis remains important in disease progression. We highlight that abiraterone metabolism and pharmacokinetics may play a role in resistance. The noncomparative design limits conclusions on the efficacy of dual therapy with abiraterone and dutasteride, but the results support development of further multifaceted approaches toward AR inhibition. <i>Clin Cancer Res; 23(4); 935–45. ©2016 AACR</i>.</p></div>

<div>Abstract<p><b>Purpose:</b> Despite the efficacy of abiraterone, a CYP17A1 inhibitor, in metastatic castration-resistant prostate cancer (CRPC), nearly all patients develop resistance. The purpose of this phase II study was to evaluate mechanisms of resistance to more complete androgen synthesis inhibition with abiraterone and dutasteride.</p><p><b>Experimental Design:</b> Eligible patients with metastatic CRPC underwent a baseline metastasis biopsy. Patients received abiraterone and prednisone for two 4-week cycles. After this time, high-dose dutasteride (3.5 mg daily) was added. Patients continued therapy until study withdrawal or radiographic progression. Repeat metastasis biopsy was obtained at progression. The primary endpoint was to assess mechanisms of resistance. Serum hormone and abiraterone levels were assessed. Tissue was assessed for androgen receptor (AR) and AR splice variant-7 (ARV7) expression.</p><p><b>Results:</b> Forty patients were enrolled. Sixty percent (<i>n</i> = 24) achieved a ≥50% reduction in prostate-specific antigen (PSA). The median time to radiographic progression was 11 months. Nearly all baseline (<i>n</i> = 29 of 31) and posttreatment (<i>n</i> = 16 of 16) tumors tested for AR nuclear expression were positive. Of those tested, ARV7 expression was present in 48% (<i>n</i> = 10 of 21) of baseline and 42% (<i>n</i> = 5 of 12) of treatment discontinuation specimens. Compared with patients with higher serum abiraterone levels at treatment discontinuation, patients with lower levels had higher circulating androgens.</p><p><b>Conclusions:</b> Despite increased androgen synthesis inhibition, we demonstrate that tumor AR axis remains important in disease progression. We highlight that abiraterone metabolism and pharmacokinetics may play a role in resistance. The noncomparative design limits conclusions on the efficacy of dual therapy with abiraterone and dutasteride, but the results support development of further multifaceted approaches toward AR inhibition. <i>Clin Cancer Res; 23(4); 935–45. ©2016 AACR</i>.</p></div>

To investigate the impact of androgen receptor splice variant 7 (AR-V7) expression on overall survival for patients with metastatic prostate cancer. The data of 113 diagnosed metastatic prostate cancer patients from January 2002 to June 2010 were collected retrospectively, including patient's age at diagnosis, prostate-specific antigen (PSA) level at diagnosis,Gleason score, clinical stage, PSA nadir during hormonal therapy, the time to PSA nadir, vital status, survival time and cause of death. The expression of AR-V7 in prostate cancer tissue was detected by using immunohistochemical staining. The correlation of AR-V7 expression and patient clinicopathological characteristics in all patients were analysed using Student t-test or Chi-square test. Cox proportional hazards regression models were used to evaluate the predictive role of AR-V7 expression and patient characteristics for overall survival. The median PSA nadir was 0.7 µg/L (ranged from 0.0 to 143.0 µg/L). The median time to PSA nadir was 8.1 months (ranged from 0.9 to 71.0 months). The follow-up was performed until March 12, 2014. During the follow-up period, 67 of 113 metastatic prostate cancer patients (59.3%) died and the median overall survival was 96 months (ranged from 5 to 135 months). The AR-V7 detection rate was 20.4% (23/113). The serum PSA level in patients with positively expression of AR-V7 was significantly higher than that without AR-V7 expression (t = 2.521, P = 0.013). Multivariate Cox regression analysis indicated that the expression of AR-V7 (HR = 2.421, P = 0.002) and time to PSA nadir (HR = 1.019, P = 0.022) were independent prognostic factors of overall survival for metastatic prostate cancer patients. The expression of AR-V7 in prostate cancer tissues and time to PSA nadir during hormonal therapy are independent prognostic factors of overall survival for metastatic prostate cancer patients. Therapy targeting AR-V7 may improve prognosis of metastatic prostate cancer patients.